Redescription and molecular characterization of two species of Pauciconfibula (Monogenea, Microcotylidae) from trachinid fishes in the Mediterranean Sea

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Redescription and molecular characterization of two species of Pauciconfibula (Monogenea, Microcotylidae)

from trachinid fishes in the Mediterranean Sea

Ramla Azizi, Chahinez Bouguerche, Mario Santoro, Delphine Gey, Fadila Tazerouti, Jean-Lou Justine, Sihem Bahri

To cite this version:

Ramla Azizi, Chahinez Bouguerche, Mario Santoro, Delphine Gey, Fadila Tazerouti, et al.. Redescrip- tion and molecular characterization of two species of Pauciconfibula (Monogenea, Microcotylidae) from trachinid fishes in the Mediterranean Sea. Parasitology Research, Springer Verlag (Germany), 2021,

�10.1007/s00436-021-07097-9�. �hal-03244165�

FISH PARASITOLOGY - ORIGINAL PAPER

Redescription and molecular characterization of two species of Pauciconfibula (Monogenea, Microcotylidae) from trachinid fishes in the Mediterranean Sea

Ramla Azizi¹_&Chahinez Bouguerche^2,3 _&Mario Santoro⁴_&Delphine Gey^5,6_&Fadila Tazerouti²_&Jean-Lou Justine³_&

Sihem Bahri¹

Received: 2 October 2020 / Accepted: 18 February 2021

#The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature 2021

Abstract

Many Pauciconfibula spp. have a long and complicated taxonomic history. The remaining unsolved taxonomic confusion in this genus is impelled by the host range and status of Pauciconfibula spp. from trachinid fishes: Pauciconfibula trachini and Pauciconfibula draconis, from Trachinus radiatus and Trachinus draco (Trachinidae), respectively. Pauciconfibula trachini was reported on Trachinus draco, type host of Pauciconfibula draconis suggesting thus a stenoxenic specificity for the former monogenean and the occurrence of two congeneric polyopisthocotyleans on a single host. Moreover, the validity of Pauciconfibula draconis was repeatedly questioned by several authors, unjustified synonymy between the two species was proposed, and the delimitations between the two species remained unsolved. Original descriptions were also incomplete and poorly illustrated. In this study, we provide a detailed illustrated redescription of both species based on newly collected specimens of Pauciconfibula trachini and Pauciconfibula draconis collected from their type hosts from off three Mediterranean localities:

Algeria, Tunisia, and Italy. Integrative taxonomy using COI sequences was applied to resolve the delimitation between Pauciconfibula trachini and P. draconis. This study provides the first DNA barcoding for members of this genus.

Keywords

Integrative taxonomy . Barcoding . COI . Pauciconfibula trachini . Pauciconfibula draconis . Trachinus

Introduction

The taxonomy of monogeneans is generally based on sclero- tized structures such as spines of the genital openings.

Pauciconfibula Dillon & Hargis, 1965 is characterized by unarmed male and female genital openings (Dillon and

Hargis

1965; Chisholm et al.1991; Cantatore, Lanfranchi &

Timi,

2011), which makes it a difficult case for taxonomists.

This genus includes six nominal species (Cantatore, Lanfranchi & Timi,

2011):

Pauciconfibula trachini (Parona

& Perugia,

1889) [syn.:

Microcotyle trachini Parona &

Perugia,

1889] on the starry weever

Trachinus radiatus

Section Editor: Shokoofeh Shamsi

* Chahinez Bouguerche

chahinezbouguerche@gmail.com

1 Research Unit of Integrative Biology and Evolutionary and Functional Ecology of Aquatic Systems, Faculty of Sciences of Tunis, Université de Tunis El Manar, Tunis, Tunisia

2 Faculté des Sciences Biologiques, Laboratoire de Biodiversité et Environnement: Interactions–Génomes, Université des Sciences et de la Technologie Houari Boumediene, BP 32, El Alia Bab Ezzouar, Algérie, Alger, Algeria

3 Institut Systématique Évolution Biodiversité (ISYEB), Muséum National d’Histoire Naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles, 57 rue Cuvier, CP 51, Paris 75005, France

4 Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Villa Comunale 1, 80121 Naples, Italy

5 Service de Systématique Moléculaire, UMS 2700 CNRS, Muséum National d’Histoire Naturelle, Sorbonne Universités, 43 Rue Cuvier, CP 26, 75231 Paris Cedex 05, France

6 UMR7245 MCAM, Muséum National d’Histoire Naturelle, 61, Rue Buffon, CP52, 75231 Paris Cedex 05, France

https://doi.org/10.1007/s00436-021-07097-9

Cuvier, 1829 off the Italian coast (Parona & Perugia,

1889, 1890);

P. draconis (Briot, 1904) [syn. : Microcotyle trachini Briot, 1904] on the greater weever T. draco Linnaeus, 1758 from the English Channel and off Scotland (Briot

1904; Nicoll 1914);

P. pogoniae (MacCallum, 1913) on the black drum Pogonias cromis (Linnaeus 1766) [syn.: M. pogoniae MacCallum, 1913] from New York, USA (MacCallum

1913);

P. euzeti (Ktari, 1971) [syn.: Aspinatrium euzeti Ktari, 1971] on the pink dentex Dentex gibbosus (Rafinesque, 1810) (Ktari

1971); and

P. gallieni (Euzet

& Ktari, 1971) [syn.: A. gallieni Euzet & Ktari, 1971] on the agujon needlefish Tylosurus acus acus (Lacépède, 1803) (Euzet and Ktari

1971) off the Tunisian coast. Except

P. gallieni collected from the inner operculum of its host (Euzet and Ktari

1971), all the previously mentioned species

inhabit gills of marine teleosts. In contrast, the latest species added to this genus, P. subsolana Chisholm, Beverley-Burton

& McAlpine, 1991, was collected in the Saint John River, Canada, from the gills of the white perch Morone americana (Gmelin, 1789) (Chisholm et al.

1991), a Moronidae Jordan &

Evermann, 1896 occurring in fresh, brackish and coastal waters.

Many Pauciconfibula spp. have a long and complicated taxonomic history (Table

1). First described as members of

Microcotyle Van Beneden and Hesse, 1863 (Briot

1904;

MacCallum

1913; Parona & Perugia, 1889, 1890) or of

Aspinatrium Yamaguti, 1963 (Euzet and Ktari

1971; Ktari 1971), they have been placed in

Bradyhaptorus Unnithan, 1971 (Unnithan

1971) then in

Pseudaspinatrium Mamaev, 1 9 8 6 ( M a m a e v

1 9 8 6

) b e f o r e t h e i r i n c l u s i o n i n Pauciconfibula (Chisholm et al.

1991; Dillon and Hargis 1965).

However, the remaining unsolved taxonomic confusion in the genus Pauciconfibula is prompted by the host range and status of Pauciconfibula spp. from trachinid fishes:

1. Pauciconfibula trachini was first described from Trachinus radiatus (Bychowsky

1961; Parona 1889,1890)

then reported on a congeneric host T. draco (Akmirza

2004;

López-Román and Guevara Pozo

1973; Radujkovic and

Euzet

1989), the type host of

P. draconis. This indicates a stenoxenic specificity for P. trachini and the occurrence of two congeneric polyopisthocotyleans on a single host.

2. The original description of P. draconis (Briot

1904) and

subsequent redescriptions (Nicoll

1914) were incomplete with

imprecise illustrations giving thus insufficient data for com- parison. Although some authors suggested the conspecificity of P. draconis with P. trachini (Chisholm et al.

1991; Dillon

and Hargis

1965; Nicoll1914), they still listed it as a distinct

species imminent additional observation (Chisholm et al.

1991; Dillon and Hargis 1965; Sproston 1946). In fact,

Dillon and Hargis (1965) clearly stated

“every effort should

be made to redescribe of these two species and re-evaluate the validity of our actions

”

(Dillon and Hargis

1965). Moreover,

in an unjustified action, Radujkovic and Euzet (1989) assigned their specimens collected on T. draco to P. trachini and synonymized the latest with P. draconis (Radujkovic and Euzet

1989).

During a parasitological survey of parasites infecting Mediterranean marine teleosts, some monogeneans were collected from the gills of two trachinids, Trachinus radiatus, and Trachinus draco. After detailed morpholog- ical study, we identified them as Pauciconfibula trachini and P. draconis (Microcotylidae). We provide here a de- tailed illustrated redescription of both species based on the newly collected specimens from their type-hosts.

Integrative taxonomy is applied herein to resolve the de- limitation between the two species.

Material and methods Collection and sampling of fish

From March 2014 to January 2018, a total of 558 trachinid fishes belonging to Trachinus draco (67 specimens) and T. radiatus (491 specimens) were collected from four marine regions, located in the Mediterranean sea: Bay of Bizerte (37°

20′ N, 9° 53′ E), Gulf of Tunis (36° 49′ N, 10° 18′ E), Sousse (35° 50′ N, 10° 38′), and Mahdia (35° 29′ N, 11° 3′ E) along the Tunisian coast; Bouharoun (36° 37

′

N, 2° 39

′

E) and Algiers (28° 01′ N, 1° 39′ E) along the Algerian coast, and Gulf of Naples (off Capri Island, 40° 33′ N, 14° 13′ E), Italy. The number of examined specimens and the range of total length (TL) according to the locality are given in Table

2. Fish specimens were transferred to the laboratory

shortly after capture and identified using keys (Fischer et al.

1987) and examined fresh on the day of purchase.

Gill arches were removed, placed in separate Petri dishes, and observed under a microscope for the presence of monogeneans.

Monogeneans

Morphological methods

Monogeneans were removed from gills using fine dissection needles, then preserved in 70% ethanol, stained with acetic carmine, dehydrated in graded ethanol series (70, 96, and 100%), cleared in clove oil, and mounted in Canada balsam.

Drawings were made with the help of a Leitz microscope

equipped with a drawing tube. Drawings were scanned and

redrawn on a computer with Adobe Illustrator. Measurements

are in micrometers and indicated as the range followed by

the mean.

Molecular methods

For a complete traceability of the molecular study, special attention was given to ensure that hosts and monogeneans were labeled with respect of host-parasite relationships (Justine et al.

2013).

Specimens of Pauciconfibula were extracted from the same host fish, and a tissue sample from the gills of the fish was taken. The extracted monogeneans were cut in three parts using a scalpel blade. Their anterior parts (which include the genital atrium) fixed in absolute ethanol then subjected to molecular analyses; posterior parts (which include the haptor) were mounted on a slide for drawing and deposition in a museum (Ayadi et al.

2017; Bouguerche et al. 2019a;

Bouguerche et al.

2019b; Bouguerche et al.2020). Nine spec-

imens were analyzed (Table

3). Slides were deposited in the

Muséum National d'Histoire Naturelle, Paris, France (MNHN), under registration numbers MNHN HEL1421- 1430.

Molecular barcoding of fish

Total genomic DNA was isolated using QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France) as per the manufacturer’s instructions. The 5

′

region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with the primers TelF1 (5′- TCGACTAATCAYAAAGAYATYGGCAC-3′) and TelR1 (5

′

-ACTTCTGGGTGNCCAAARAATCARAA-3

′

)

(Dettaï et al.

2011). PCR reactions were performed in 20μ

l, containing 1 ng of DNA, 1× CoralLoad PCR buffer, 3 mM MgCl2, 66

μM of each dNTP, 0.15μM of each primer, and

0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94 °C, followed by 40 cycles at 94 °C for 30 s, 48 °C for 40 sec, and 72 °C for 50 s, with a final extension at 72 °C for 7 min. PCR products were purified (Ampure XP Kit, Beckman Coulter) and sequenced in both directions on a 3730xl DNA Analyzer 96-capillary sequencer (Applied Biosystems, Foster City, USA). We used the CodonCode Aligner version 3.7.1 software (Codon Code Corporation, Dedham, MA, USA) to edit sequences, compared them to the GenBank database content with BLAST, and deposited them in GenBank under accession numbers MW484937- MW484939.

Species identification was confirmed with the BOLD identifica- tion engine (Ratnasingham and Hebert

2007).

COI sequences of monogeneans

Total genomic DNA was isolated using QIAmp DNA Micro Kit (Qiagen). The specific primers JB3 (=COIASmit1) (for- ward 5

′

-TTTTTTGGGCATCCTGAGGTTTAT-3

′

) and JB4.5 (=COI-ASmit2) (reverse 5′-TAAAGAAAGAACAT AATGAAAATG-3′) were used to amplify a fragment of 424 bp of the COI gene (Bowles et al.

1995; Littlewood

et al.

1997). PCR reaction was performed in 20μl, containing

1 ng of DNA, 5× iProof HF buffer, 0.25 mM dNTP, 0.15

μM

of each primer, and 0.5 units of iProof HF DNA polymerase

Table 1 Synonyms, hosts, and localities ofPauciconfibulaspp. in the literature. *junior synonym

Parasites Synonyms Type-host Type-locality Source

Pauciconfibula trachini (Parona & Perugia,1889)

Dillon & Hargis, 1965

Microcotyle trachini* Parona & Perugia,1889 Diplasiocotyle trachini* (Parona & Perugia,

1889) Tripathi, 1954

Aspinatrium trachini* (Parona & Perugia, 1889) Yamaguti, 1963

Bradyhaptorus trachini* (Parona & Perugia, 1889) Unnithan, 1971

Trachinus radiatus (Trachinidae)

Naples, Italy, Mediterranean

(Parona & Perugia,1889,1890;

Tripathi1956; Unnithan1971;

Yamaguti1963)

Pauciconfibula draconis (Briot, 1904) Dillon & Hargis,

1965

Microcotyle draconis* (Briot, 1904) Bradyhaptorus draconis* (Briot,

1904) Unnithan, 1971

Pseudoaspinatrium draconis* (Briot, 1904) Mamaev, 1986

Trachinus draco (Trachinidae)

North Sea, North Atlantic

(Briot1904; Dillon and Hargis1965;

Unnithan1971) Briot (1904) ; Dillon and Hargis (1965)

Pauciconfibula pogoniae (MacCallum, 1913) Chisholm,

Beverley-Burton &

McAlpine,1991

Pseudoaspinatrium pogoniae*

(MacCallum, 1913) Mamaev, 1986

Pogonias cromis

(Linnaeus, 1766) (Sciaenidae)

New York, North Atlantic

(Chisholm et al.1991; MacCallum 1913; Mamaev1986)

Aspinatrium euzeti*

Ktari, 1971

Pseudoaspinatrium euzeti* (Ktari, 1971) Mamaev, 1986

Pauciconfibula euzeti(Ktari, 1971) Chisholm, Beverley-Burton & McAlpine,1991

Dentex gibbosus (Rafinesque, 1810) (Sparidae)

Tunisia, Mediterranean (Chisholm et al.1991; Ktari1971;

Mamaev1986)

Pauciconfibula subsolana Chisholm, Beverley-Burton &

McAlpine, 1991

- Morone americana(Gmelin,

1789) (Moronidae)

Canada, freshwater (Chisholm et al.1991)

Pauciconfibula patagonensis Cantatore, Lanfranchi & Timi,

2011

- Congiopodus peruvianus

(Cuvier, 1829) (Congiopodidae)

Argentina, Atlantic (Cantatore, Lanfranchi & Timi,2011)

(Biorad). Thermocycles consisted of an initial denaturation step at 94 °C for 2 min, followed by 37 cycles of denaturation at 94 °C for 30 s, annealing at 48 °C for 40 s, and extension at 72 °C for 50 s. The final extension was conducted at 72 °C for 5 min. Sequences were edited with CodonCode Aligner soft- ware version 3.7.1 (CodonCode Corporation, Dedham, MA, USA), compared to the GenBank database content with BLAST, and deposited in GenBank under accession numbers MW484928- MW484936.

Trees and distances

Most sequences of Microcotylidae available in GenBank were included in the phylogenetic analysis (Table

4), with 2

Pauciconfibula draconis and 7 P. trachini sequences obtained in the present study (2 off the Algerian coast, 4 off the Tunisian coast, and 1 from Gulf of Naples, Italy). Sequences of Allogastrocotyle bivaginalis Nasir and Fuentes Zambrano, 1983 (Gastrocotylidae Price, 1943) and Kuhnia scombri (Kuhn, 1829) (Mazocraeidae Price, 1936) were used as outgroup. Phylogenetic analyses were performed in MEGA ver- sion 7 (Kumar et al.

2016) based on the best scoring. Maximum

likelihood was used for the best fitting tree according to the Tamura-Nei (TN93), rates among sites were gamma distributed (G); the number of discrete gamma categories was 5. The robust- ness of the inferred analysis was assessed using a bootstrap pro- cedure with 1 000 replications. Genetic distances (p-distance and

Kimura-2 parameter distance (Kimura

1980) were estimated

with MEGA7 and all codon positions were used.

Results

Molecular identification of fish

The provisional identification of the two fish species using morphological characteristics was confirmed by DNA barcoding approach. BLAST analysis of the COI sequences of fish species of the present study with NCBI and BOLD database showed sequence similarity values of 99.85% for Trachinus draco and T. radiatus, respectively.

Molecular characterization of monogeneans

The COI sequences of Pauciconfibula spp. were aligned with several microcotylid. For trees, the neighbor-joining and max- imum likelihood methods led to similar topologies; we show only the later in Fig.

1. The analysis involved 28 nucleotide

sequences, and there was a total of 263 positions in the final dataset.

Microcotylidae clustered in the same clade, distinct from the outgroup. All sequences of Pauciconfibula trachini and P. draconis generated in the present study nested within a monophyletic lineage. Pauciconfibula trachini from the type

Table 2 Number and range of

total length (TL) of the examined hosts according to the locality

Species Locality Number Range of TL (mm)

T. draco Bay of Bizerte (Tunisia) 190 21.5–30.5

Sousse (Tunisia) 110 15.5–28.5

Mahdia (Tunisia) 156 19.5–29.5

Gulf of Tunis (Tunisia) 65 15.5–23.5

Bouharoune (Algeria) 134 18–32.5

T. radiatus Bay of Bizerte (Tunisia) 26 19.5–34.5

Sousse (Tunisia) 18 14.5–31.5

Mahdia (Tunisia) 46 14.5–34.5

Algiers (Algeria) 1 40

Off Capri Island, Gulf of Naples (Italy) 1 31

Table 3 Fish, Monogeneans, and their COI sequences. To ensure full traceability and respect of host-parasite relationships, one monogenean was collected from one fish and each fish and monogenean individuals was sequenced

Fish species Fish Id Fish COI sequence Monogenean species Locality Monogenean Id Monogenean COI sequence

Voucher deposited in MNHN

Trachinus draco Trdr Br1 MW484937 Pauciconfibula draconis Algeria Trdr Br1 MO-01 MW484928 HEL1421

Trachinus draco Trdr Br2 MW484938 Pauciconfibula draconis Algeria Trdr Br1 MO-02 MW484929 HEL1422

Trachinus radiatus TrraRamla1 MW484939 - - - - -

Trachinus radiatus - - Pauciconfibula trachini Algeria TrraAl MO-01 MW484930 HEL1423

Trachinus radiatus - - Pauciconfibula trachini Algeria TrraAl MO-03 MW484931 HEL1424

Trachinus radiatus - - Pauciconfibula trachini Italy Trra MO-08 MW484935 HEL1425

Trachinus radiatus - - Pauciconfibula trachini Tunisia Trra MO-01 MW484932 HEL1426

Trachinus radiatus - - Pauciconfibula trachini Tunisia Trra MO-02 MW484933 HEL1427

Trachinus radiatus - - Pauciconfibula trachini Tunisia Trra MO-04 MW484934 HEL1428

Trachinus radiatus - - Pauciconfibula trachini Tunisia TraraxBrx MW484927 HEL 1429

Trachinus radiatus - - Pauciconfibula trachini Tunisia TrraRa1Pau1 MW484936 HEL1430

host Trachinus radiatus off three Mediterranean localities (Algeria, Tunisia, and Italy) clustered in a robust clade (100 bootstrap) whereas P. draconis from the type host T. draco clustered in a distinct and well-supported clade (100 boot- strap), supporting thus P. trachini and P. draconis as distinct species. The support value of other clades was generally low.

Distances were computed using p-distance and Kimura 2- parameter distance (Table

5). The differences were minor so

only p-distances are commented here. Sequences of Pauciconfibula draconis from the type-host Trachinus draco off the Algerian coast were identical between them (0% intra- specific variation). Sequences of P. trachini from the type host T. radiatus off three Mediterranean localities (Algeria,

Tunisia, and Italy) showed little to no variations: 0-2%, an order of magnitude inferior to the interspecific distances.

Pauciconfibula trachini (Parona & Perugia, 1889) Dillon & Hargis, 1965 (Figs. 2, 3, and 4)

Synonymes: Microcotyle trachini Parona & Perugia, 1889 (Parona & Perugia,

1889, 1890);

Diplasiocotyle trachini (Parona & Perugia, 1889) Tripathi, 1956 (Tripathi

1956);

Aspinatrium trachini (Parona & Perugia,

1889) Yamaguti,

1963 (Yamaguti

1963);

Bradyhaptorus trachini (Parona &

Perugia,

1889) Unnithan, 1971 (Unnithan1971).

Table 4 Sequences used in the present molecular study

Parasite species Host species Origin GenBank Source

Pauciconfibula draconis Trachinus draco Algeria MW484928 Present study

MW484929

Pauciconfibula trachini Trachinus radiatus Algeria MW484930

MW484931 Tunisia MW484932 MW484933 MW484934 MW484927 MW484936

Italy MW484935

Microcotyle visa

Bouguerche, Gey, Justine and Tazerouti, 2019

Pagrus caeruleostictus Algeria MK275652 (Bouguerche et al.2019a) MK275653

MK275654 Microcotyle isyebi

Bouguerche, Gey, Justine and Tazerouti, 2019

Boops boops Algeria MK317922 (Bouguerche et al.2019b) Spain MN816019 (Víllora-Montero et al.2020) Microcotyle algeriensis

Ayadi, Gey, Justine and Tazerouti, 2017

Scorpaena notata Algeria KX926443 (Ayadi et al.2017) KX926444

Microcotylesp. Helicolenus dactylopterus Algeria KX926446 (Ayadi et al.2017)

KX926447 Microcotyle sebastis

Goto, 1894

Sebastes schlegeli South Korea NC009055 (Park et al.2007) DQ412044

Microcotyle erythrini van Beneden and Hesse, 1863

Pagellus erythrinus France AY009159 (Jovelin and Justine2001) Spain MN816012 (Víllora-Montero et al.2020)

MN816013 M. whittingtoni

Víllora-Montero et al.,2020

Dentex dentex Spain MN816010 (Víllora-Montero et al.2020) MN816011

M. caudata Goto, 1894

Sebastes inermis Inland Sea LC472527 Kamio and Ono (unpublished data) LC472528

Microcotylesp. Helicolenus dactylopterus Algeria KX926446 (Ayadi et al.2017)

KX926447

Microcotylesp. Sebastiscus marmoratus Inland Sea LC472526 Kamio and Ono (unpublished data)

Kuhnia scombri(Kuhn, 1829) Scomber japonicus China KU380119 (Yan et al.2016)

Paramicrocotylesp. Pinguipes chilensis Chile KJ794215 (Oliva et al.2014)

Pedocotyle bravoiLuque-Alejos and Iannacone-Oliver, 1990 Prolatilus jugularis Chile KJ794211 (Oliva et al.2014) Sparicotyle chrysophryivan Beneden and Hesse, 1863 Boops boops Adriatic GQ240258 (Mladineo et al.2009)

Sparicotyle chrysophryii Sparus aurata Adriatic GQ240252 (Mladineo et al.2009)

Pagellicotyle mormyri (Lorenz, 1878)

Lithognathus mormyrus France AY009160 (Jovelin and Justine2001)

Pyragraphorus hollisae Euzet and Ktari, 1970

Trachinotus ovatus France AY009162 (Jovelin and Justine2001)

Bivagina pagrosomi (Murray, 1931)

Sparus aurata Australia Z83003 (Littlewood et al.1997)

Pagellicotyle mormyri Lithognathus mormyrus France AY009160 (Jovelin and Justine2001)

Type-host: Trachinus radiatus Cuvier, 1829 (Trachinidae), starry weever (Parona & Perugia,

1889,1890).

Type-locality: off Genova, Italy, Mediterranean (Parona &

Perugia,

1889,1890).

Additional localities: off Naples, Italy, this paper;

Bouharoun, off the Algerian coast, this paper; Tunisia, this paper.

Site on host: Gills.

Specimens from Algeria: Vouchers deposited in the collec- tions of the Muséum National d’Histoire Naturelle, Paris (MNHN HEL1371 - HEL1393, MNHN HEL1410

–

HEL1420).

Specimens from Tunisia: Vouchers deposited in the collec- tions of the Muséum National d’Histoire Naturelle, Paris (MNHN HEL1394–HEL1396).

Vouchers with molecular information: posterior parts of specimens containing the haptor mounted on slide, anterior part used for molecular analysis; off the Algerian coast (MNHN HEL1423, GenBank MW484930; MNHN HEL1424, GenBank MW484931), from Italy (MNHN HEL1425, GenBank MW484935, from Tunisian waters (MNHN HEL1426, GenBank MW484932; MNHN HEL1427, GenBank MW484933; MNHN HEL1428, GenBank MW484934; MNHN HEL1429, GenBank MW484927; MNHN HEL1430, GenBank MW484936).

Description

Body elongated, slightly thinned in its anterior region (Fig.

2a). Haptor symmetrical and quadrangular, armed

with 8 to 10 pairs of clamps arranged symmetrically in two parallel rows. Clamps of unequal sizes: first pair always smaller.

Clamps of Microcotyle type (Fig.

2d). Ventral arm of me-

dian spring a1 elongated. Distal part of a1 T-shaped. Ventral arm of ventral jaw b1 arched (Fig.

4a). Dorsal arms of median

spring a2 and a3 with two parallel rows of small pores of different shape and size. Dorsal arm of ventral jaw b2 long.

Dorsal arm of dorsal jaw c arched (Fig.

4b). On proximal and

distal side, a2 and b2 connected by a muscle; b1 and c surrounded by a muscle (Fig.

2d). We note the presence of

numerous lateral epidermal thickenings (Fig.

4c).

Anterior extremity rounded. Prohaptoral suckers paired, septate, with numerous minute spines. Pharynx median, volu- minous, (Fig.

2b) Caeca with numerous lateral and axial di-

verticula; caeca obscured by vitellarium in most body parts.

Testes numerous, in intercæcal field of posterior third. Vas deferens wide, conspicuous, running forward along body mid- line to genital atrium. Genital atrium middorsal, muscular and unarmed (Fig.

2b).

Ovary pre-testicular begins on the right side of the body. It makes a handle and passes on the left by a thin elbow (Fig.

3). It goes up on the midline, makes a

second loop to return to the right, where it widens

before emerging into the oviduct. Oviduct dorsal, connecting common vitelline duct and uterus. Uterus descending and then ascending, running forward along body midline dorsal to vas deferens.

Vitellarium follicular, co-extensive with caeca, ex- tending from level of genital atrium to the haptor.

Posterior extremities of vitelline fields symmetrical often joined. Vitelloducts Y-shaped with very long lateral branches. Dorsal transverse vitelloducts fused at level of ovary. Common vitelline duct median and dorsal to ovary, fairly long. Eggs fusiform with two polar fila- ments: anterior filament long and curled; posterior fila- ment short and thick (Fig.

2c).

Pauciconfibula draconis (Briot, 1904) Dillon & Hargis, 1965 (Figs. 5, 6, and 7)

Synonymes: Microcotyle draconis Briot, 1904 (Briot

1904);

Bradyhaptorus draconis (Briot, 1904) Unnithan, 1971 (Unnithan

1971);

Pseudoaspinatrium draconis (Briot, 1904) Mamaev, 1986 (Mamaev

1986).

Type-host: Trachinus draco, Linnaeus 1758 (Trachinidae), greater weever (Briot

1904).

Type-locality: English Channel, North Sea, Atlantic (Briot

1904).

Additional localities: Spain (López-Román and Guevara Pozo

1973); Montenegro (Radujkovic and Euzet 1989);

Turkey (Akmirza

2004); off the Algerian coast, Bouharoun,

this paper.

Site on host: Gills.

Specimens from Algeria: Vouchers deposited in the collec- tions of the Muséum National d’Histoire Naturelle, Paris (MNHN HEL1397

–

HEL1409).

Vouchers with molecular information posterior parts of specimens containing the haptor mounted on slide, anterior part used for molecular analysis; off the Algerian coast (MNHN HEL1421, GenBank MW484928, MNHN HEL1422, GenBank MW484929).

Description

Body stocky (Fig.

5a). Haptor quadrangular armed with 8 to

10 pairs of clamps arranged in two symmetrical rows; clamps rows spaced apart; clamps size regularly decreasing antero- posteriorly. Clamps Microcotyle type (Fig.

5e), disposition of

sclerites is identical to that of Pauciconfibula trachini (Fig.

7a, b). We also note the presence of numerous lateral epider-

mal thickenings (Fig.

7c).

Prohaptoral suckers paired, globular, opening laterally and

surrounded by numerous papillae (Fig. b). Dorsally, cavity of

each buccal organ is divided by transverse partition in two

unequal compartments. Pharynx bulky. Intestinal caeca with

numerous lateral diverticula.

Testes variously shaped and sized, occupying the entire intercaecal field of posterior quarter of body. Vas deferens dorsal, broad, and conspicuous, running forward along body midline to genital atrium. Genital atrium anterior, midventral (Fig.

5c).

Ovary pre-testicular, oblong, and large. Ovary begins on left side of body by an elongated mass (Fig.

6). It goes up to

the left then passes to the right and dorsally descends to the immature part. It ascends, then passes again to the left where it widens before emerging into oviduct. Genito-intestinal canal detaching from oviduct and abutting ventrally in left intestinal caecum. Uterus dorsal, running forward along body midline to genital atrium.

Vitellarium follicular, surrounding laterally intestinal caeca;

vitellarium extending from level of genital atrium to the haptor.

Posterior extremities of vitelline fields symmetrical, fused

posteriorly. Vitelloducts Y-shaped with very long lateral branches. Dorsal transverse vitelloducts fused at ovary.

Common vitelline duct median and dorsal to ovary, long.

Eggs oval and elongated, with two polar filaments: posterior filament shorter and ends in a claw (Fig.

5d).

Comparison

In the present study, we compared measurements and counts of Pauciconfibula trachini from its type host Trachinus radiatus, off two Mediterranean localities, Tunisia and Algeria: P. trachini from Algeria and Tunisia are identical, with an overlap in morphometric data (Table

6).

Morphometric data of Pauciconfibula trachini off Algeria and Tunisia were within the range of the original description (Parona & Perugia,

1889, 1890) [in the following section, Fig. 1 Molecular phylogenetic analysis by the Maximum Likelihood

method based on the Hasegawa-Kishino-Yano model [1] of COI se- quences of monogeneans. Bootstraps percentages (1000 replicates) are

indicated next to the branches when significant. There was a total of 263 positions in the final dataset. The Neighbor-Joining tree had similar to- pology and is not represented

Table5Estimateofevolutionarydivergencebetweensequences.Therewasatotalof263positionsinthedataset.Distancesarep-distances,shownaspercentages 23456789101112131415161718192021222324252627282930 MicrocotylewhittingtoniexDentexdentex MicrocotylewhittingtoniexDentexdentex1 MicrocotyleerythriniexPagelluserythrinus1211 MicrocotyleerythriniexPagelluserythrinus13122 MicrocotylecaudataexSebastes15151718 MicrocotylecaudataexSebastesinermis141416171 MicrocotyleisyebiexBoopsboops141415141111 MicrocotyleisyebiexBoopsboops1515141411112 MicrocotylevisaexPagruscaeruleosticus121213139987 MicrocotylevisaexPagruscaeruleosticus1212131410101092 MicrocotylealgeriensisexScorpaenanotata17161717141413131314 MicrocotylealgeriensisexScorpaenanotata171617171414131313140 MicrocotylesebastisexSebastesschlegeli16161417101010810111313 PolylabrishalichoeresexHalichoeresnigrescens21212324242423232222232323 Mormyrocotylemormyriex Lithognathusmormyrus1920202018172120181919191522 SparicotylechrysophryiiexSparusaurata212119192324242424242323212416 SparicotylechrysophryiiexBoopsboops2222202024242424242424242226185 KuhniascombriexScomberjaponicus2626252526262627272827272624212728 PauciconfibuladraconisexT.dracoAlgeria232222222524232324242222222722252728 PauciconfibuladraconisexT.dracoAlgeria2322222225242323242422222227222527280 PauciconfibulatrachiniexT.radiatusAlgeria23232122242325252527232322272124262788 PauciconfibulatrachiniexT.radiatusAlgeria242321222323242425262222222720242626882 PauciconfibulatrachiniexT.radiatusTunisia2423222224222424252622222227212427258812 PauciconfibulatrachiniexT.radiatusTunisia24232222242224242526222222272124272588120 PauciconfibulatrachiniexT.radiatusTunisia242322222422242425262222222721242726881211 PauciconfibulatrachiniexT.radiatusTunisia2323212124222424252622222227202426268812000 PauciconfibulatrachiniexT.radiatusItaly23222121222224232425222221272023252699222221 PauciconfibulatrachiniexT.radiatusTunisia242322222422242425262222222721242725881200102 Allogastrocotylebivaginalis27272928262623232525282827292630302129292928282828282928 Bivaginapagrosomi1919222322222021212220202224222022252424242424242424242426

compared measurements are giving as follow: from Tunisia, from Algeria vs. off type locality]; especially with regard of body width (900 (700−1100), 541 (364− 825) vs. 500); prohaptoral suckers and pharynx size; clamps number (8−10, 6−10 pairs vs. 8

−10) and size; testes number (39−53, 34−58 vs. 30), and eggs

size (257 (250−270) ×79 (62−97), 261 ( 240

−

285) ×75 (70

−

80) vs. 280 × 80). We noted than specimens from off the Italian coast were remarkably larger (4100 (3700−4800), 4082 (2244

−

5595) vs. 9000−11000).

Measurements and counts of Pauciconfibula draconis agreed with the original description (Briot

1904) and the subsequent

redescription (Nicoll

1914), in regard of body length (2428

(1652

−2944 vs. 5000) and clamps number (11−15 vs. 5−11).

Pauciconfibula draconis differs from P. trachini off the Algerian coast by having a smaller pharynx (64

−

122 × 61

−

114 vs. 137

−

190 × 130

−

178) and a smaller genital atrium (44

−

81 × 48

−

65 vs. 58

–

105 × 63

–

105); by length of anterior (40

−

46 vs. 47− 90), median (50−71 vs. 64− 102) and posterior (40

−

54 vs. 61− 85) clamps; by having more clamps (11−15 pairs vs. 6−10 pairs) and fewer testes (20−28 vs. 34−58).

Discussion

In the present study, we compared measurements and counts of Pauciconfibula trachini from their type host Trachinus

Fig. 2 Pauciconfibula trachini

fromTrachinus radiatus.A Whole body, MNHN HEL1412.

BAnterior extremity showing relative positions of prohaptoral suckers, pharynx, and male copulatory organ, MNHN HEL1412.CEgg.DClamp (dorsal view), MNHN HEL1411

Fig. 3 Pauciconfibula trachini fromTrachinus radiatus. Detail of anatomy of reproductive organs in the region of ovary, MNHN HEL1412

Fig. 4 Pauciconfibula trachinifromTrachinus radiatus. Clamp:Aventral jaw,Bdorsal jaw,Cclamp (dorsal view), MNHN HEL1411

radiatus, off two Mediterranean localities, Tunisia and Algeria, and there was an apparent overlap. Specimens de- scribed in the original description were larger. However, it is well known that body length is the least reliable feature for species delimitation in monogeneans (Rohde and Watson

1985).

COI sequences of Pauciconfibula trachini off the three Mediterranean localities, Algeria, Tunisia, and Italy differed by only 0−2%. Differences lower than 4% in COI sequences are commonly read as intraspecific variations (Aquaro et al.

2012; Hansen et al.2007; Vanhove et al. 2013; Ward et al.

2005). Differences ranging from 0.2−5.6% in COI sequences

of polyopisthocotylean monogeneans were reported as

intraspecific divergence (Bouguerche et al.

2019a). We thus

consider that all specimens belong to the same species.

Pauciconfibula draconis was distinguished from P. trachini off the Algerian coast by the size of pharynx, genital atrium, clamps; number of clamps and testes. They differed also by their body shape, with P. draconis having an evident stocky appearance. However, we note that the number of studied specimens was low, and measurements and counts overlapped.

Most importantly, COI sequences of Pauciconfibula trachini from Trachinus radiatus differed from those of P. draconis from T. draco by 8−9%, a divergence higher than 4% threshold in COI sequences of Monogenea (Aquaro et al.

Fig. 5 Pauciconfibula draconis fromTrachinus draco.AWhole body, MNHN HEL1404.B Anterior extremity showing relative positions of prohaptoral suckers and pharynx, MNHN HEL1404.CGenital atrium, MNHN HEL1409.DEgg, MNHN HEL1408.EClamp, MNHN HEL1409 (ventral view), MNHN HELxxx

2012) and other reported intraspecific variations (Bouguerche

et al.

2019a). The 8−9 % difference reported here do not fall

within the intraspecific range and thus we consider that

P. trachini from T. radiatus and P. draconis from T. draco are regarded as different and distinct species. We thus con- firm, for the first time with molecular evidence, delimitation

Fig. 6 Pauciconfibula draconis

fromTrachinus draco. Detail of anatomy of reproductive organs in the region of ovary, MNHN HEL1404.

Fig. 7 Pauciconfibula draconisfromTrachinus draco. Clamp:Adorsal jaw,Bventral jaw,CClamp (dorsal view), MNHN HEL1409.

Table6MeasurementsofPauciconfibulatrachinifromvariouslocalities.-:notstated.*Diameter. ParasitesPauciconfibulatrachiniPauciconfibuladraconis HostsTrachinusradiatusTrachinusdraco PreviousidentificationMicrocotyletrachini//MicrocotyledraconisAspinatriumtrachini/ LocalityItaly,MediterraneanAlgeria, Mediterrane- an Tunisia, Mediterrane- an EnglishChannel, AtlanticScotland, AtlanticSpain,MediterraneanMontenegro, MediterraneanAegeanSea, MediterraneanAlgeria, Mediterrane- an Numberofspecimens-11,mature16,mature>302, imma- ture

1,mature42910,mature Source(ParonaandPerugia 1889;1890)PresentstudyPresentstudy(Briot 1904)(Nicoll1914)(López-RománandGuevara Pozo1973)(Radujkovicand Euzet1989)(Akmirza2004)Presentstudy Bodylength(9000–11000)3205(1010– 4465)3102(1120– 3796)5000--3680–43002800–40002700–41002428(1652− 2944) Haptorlength-783(343– 1190)900(800− 1100)---1000–1300582(343−790) Totallength-4082(2244– 5595)4100(3700− 4800)---3355(2244− 6621) Bodywidth500541(364−825)900(700− 1100)1300--860–1030650620–790793(364−915) Prohaptoralsuckers length56*63(41−73)64(60−68)*---63–7753*49(35−58) Prohaptoralsuckers width67(46−77)---63–6950(32−60) Pharynxlength98*157(137–190)139(120−160) *---88–99*--100(64−122) Pharynxwidth146(130–178)---97(61−114) Clampsnumber(ofpairs)(8–10)8(6–10)8(7−10)571113–168–109–1313(11−15) ClamplengthLargest:>126* Smallest:4264(47−90)112(100−120)---83–10080–9842(40−46) 90(64−102)57(50−71) 78(61−85)49(40−54) Clampwidth61(40−84)8170−95---70–9067–729038(35−40) 80(66−93)42(35−48) 68(47−80)41(35−54) Genitalatriumlength-82(58–105)---60(44−81) Genitalatriumwidth-84(63–105)---56(48−65) Numberoftestes3047(34–58)48(39–53)--5050–5550–5523(20−28) Distancegenital atrium−anteriorend-360(290–430)466(410−520)---300–400--277(210−365) Egglength80261(240–285)257(250−270)---280–316--260(223−311) Eggwidth2875(70–80)79(62−97)---89–116--68(57−85)

between the two species and the validity of P. draconis. The synonymy between P. trachini and P. draconis (Radujkovic and Euzet

1989) was unjustified.

The remaining puzzling situation regards the host range of Pauciconfibula trachini (Table

7). The mention of this mono-

genean on Trachinus draco (Akmirza

2004; López-Román

and Guevara Pozo

1973; Radujkovic and Euzet1989), type

host of P. draconis advocate a stenoxenic specificity for P. trachini, and the occurrence of two congeneric polyopisthocotyleans, on a single host.

However, our careful comparison of specimens of Pauciconfibula assigned to P. trachini; and collected from Trachinus draco off the Aegean Sea (Akmirza

2004),

Montenegro (Radujkovic and Euzet

1989), and Spain

(López-Román and Guevara Pozo

1973) with the microtylid

assigned to this host P. draconis (Briot

1904; Nicoll 1914)

showed an overlap in all measurements and counts (Table

6). Most interestingly, the stocky body appearance of

Pauciconfibula cf. trachini reported on T. draco (Akmirza

2004; López-Román and Guevara Pozo 1973; Radujkovic

and Euzet

1989) matched that of

P. draconis (P. trachini being rather more elongated). We suggest that previous mentions of P. trachini on T. draco (Akmirza

2004; López-Román and

Guevara Pozo

1973; Radujkovic and Euzet1989) were in fact

misidentifications of P. draconis. Hence, new host records and considerations of generalist polyopisthocotyleans are to be

considered with caution and should be based imperatively on integrative taxonomy.

Acknowledgements Our thanks are also due to fishermen from Algiers and especially Bourouba Mohamed, Boumerahe Malak, and Ammi Said.

Funding This study was supported by Institut de Systématique, Évolution, Biodiversité (ISYEB), Muséum national d’Histoire naturelle (MNHN) Paris, France), and a framework agreement project of the DeepBlue Project: Distance Crossborder Traineeship Programmeco- financed by“The European Maritime and Fisheries Fund (EMFF).” The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Declarations

Competing interests The authors declare no competing interests.

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